The present invention is concerned with a class of polymer precursors with narrow molecular weight distribution and the production therefrom of physiologically soluble polymer therapeutics, functionalised polymers, pharmaceutical compositions and materials, all with similar molecular weight characteristics and a narrow molecular weight distribution.
Polymer Therapeutics (Duncan R: Polymer therapeutics for tumour specific delivery Chem and Ind 1997, 7, 262-264) are developed for biomedical applications requiring physiologically soluble polymers and include biologically active polymers, polymer-drug conjugates, polymer-protein conjugates, and other covalent constructs of polymer with bioactive molecules. An exemplary class of a polymer-drug conjugate is derived from copolymers of hydroxypropyl methacrylamide (HPMA) which have been extensively studied for the conjugation of cytotoxic drugs for cancer chemotherapy (Duncan R: Drug-polymer conjugates: potential for improved chemotherapy. Anti-Cancer Drugs, 1992, 3, 175-210. Putnam D, Kopecek J: Polymer conjugates with anticancer activity. Adv.Polym.Sci., 1995, 122, 55-123. Duncan R, Dimitrijevic S, Evagorou E: The role of polymer conjugates in the diagnosis and treatment of cancer. STP Pharma, 1996, 6, 237-263). An HPMA copolymer conjugated to doxorubicin known as PK-1, is currently in Phase II evaluation in the UK. PK-1 displayed reduced toxicity compared to free doxorubicin in the Phase I studies (Vasey P, Twelves C, Kaye S, Wilson P, Morrison R, Duncan R, Thomson A, Hilditch T, Murray T, Burtles S, Cassidy J: Phase I clinical and pharmacokinetic study of PKI (HPMA copolymer doxorubicin): first member of a new class of chemotherapeutic agents: drug-polymer conjugates. Clin. Cancer Res., 1999, 5, 83-94). The maximum tolerated dose of PK-1 was 320 mg/m2 which is 4-5 times higher than the usual clinical dose of free doxorubicin.
The polymers used to develop Polymer Therapeutics may also be separately developed for other biomedical applications where the polymer conjugate is developed (e.g. as a block copolymer) to form aggregates such as polymeric micelles and complexes (Kataoka K, Kwon G, Yokoyama M, Okano T. Sakurai Y: Block copolymer micelles as vehicles for drug delivery. J. Cont.Rel., 1993, 24, 119-132. Inoue T, Chen G, Nakamae K, Hoffman A: An AB block copolymer of oligo(methyl methacrylate) and poly(acrylic acid) for micellar delivery of hydrophobic drugs. J Cont. Rel., 1998, 51, 221-229. Kwon G, Okano T: Polymeric micelles as new drug carriers. Adv. Drug Del. Rev., 1996, 21, 107-116.). The polymers used to develop Polymer Therapeutics may also be separately developed for other biomedical applications that require the polymer be used as a material rather than as a physiologically soluble molecule. Thus, drug release matrices (including microspheres and nanoparticles), hydrogels (including injectable gels and viscious solutions) and hybrid systems (e.g. liposomes with conjugated poly(ethylene glycol) (PEG) on the outer surface) and devices (including rods, pellets, capsules, films, gels) can be fabricated for tissue or site specific drug delivery. Polymers are also clinically widely used as excipients in drug formulation. Within these three broad application areas: (1) physiologically soluble molecules, (2) materials and (3) excipients, biomedical polymers provide a broad technology platform for optimising the efficacy of a therapeutic bioactive agent.
Therapeutic bioactive agents which can be covalently conjugated to a polymer include a drug, peptide and protein. Such conjugation to a soluble, biocompatible polymer can result in improved efficacy of the therapeutic agent. Compared to the free, unconjugated bioactive agent, therapeutic polymeric conjugates can exhibit this improvement in efficacy for the following main reasons: (1) altered biodistribution, (2) prolonged circulation, (3) release of the bioactive in the proteolytic and acidic environment of the secondary lysosome after cellular uptake of the conjugate by pinocytosis and (4) more favourable physicochemical properties imparted to the drug due to the characteristics of large molecules (e.g. increased drug solubility in biological fluids) (Note references in Brocchini S and Duncan R: Polymer drug conjugates: drug release from pendent linkers. The Encyclopedia of Controlled Drug Delivery, Wiley, N.Y., 1999, 786-816.).
Additionally, the covalent conjugation of bioactive agents to a polymer can lead to improved efficacy that is derived from the multiple interactions of one or more of the conjugated bioactive agents with one or more biological targets. Such polyvalent interactions between multiple proteins and ligands are prevalent in biological systems (e.g. adhesion of influenza virus) and can involve interactions that occur at cell surfaces (e.g. receptors and receptor clusters) (Mammen M, Choi S, Whitesides GM: Polyvalent interactions in biological systems: Implications for design and use of multivalent ligands and inhibitors. Angew. Chem. Int. Ed. 1998, 37, 2754-2794. Whitesides G, Tananbaum JB. Griffin J, Mammen M: Molecules presenting a multitude of active moieties. PCT Int. Appl. WO 9846270). Multiple simultaneous interactions of a polymer bioactive conjugate will have unique collective properties that differ from properties displayed by the separate, individual, unconjugated bioactive components of the conjugate interacting monovalently.
Additionally, an appropriately functionalised polymer can interact with mucosal membranes (e.g. in the gastrointestinal, respiratory or vaginal tracts) by polyvalent interactions. Such a property is valuable for prolonged and/or preferential localisation of a functionalised polymeric excipient used for site specific delivery or altering optimally the biodistribution of a bioactive agent.
Additionally polymer bioactive agent conjugates and/or aggregates can be designed to be stimuli responsive (Hoffman A, Stayton PS: Interactive molecular conjugates. U.S. Pat. No. 5,998,588), for example, to be for membranelytic after being taken up by a cell by endocytosis. These polymeric constructs must incorporate the membrane penetration features seen in natural macromolecules (toxins and transport proteins) and viruses. Cytosolic access has been shown to be rate limiting during polymer-mediated transfection (Kichler A, Mechtler A, Mechtler K, Behr JP, Wagner E: Influence of membrane-active peptides on lipospermine/DNA complex mediated gene transfer, Bioconjugate Chem., 1997, 8(2), 213-221.). Many of the cationic polymers (e.g. (poly-L-lysine) (PLL) and poly(ethyleneimine) (PEI), chitosan and cationic PAMAM dendrimers) that have been used for in vitro transfection studies are either cytotoxic (IC50 values  less than 50 xcexcg/ml) or hepatotropic after i.v. injection. Such molecules are totally unsuitable for in vivo/clinical development. Alternative endosomolytic molecules have been proposed but are either too toxic (i.e. poly(ethylenimine) or potentially immunogenic (e.g. fusogenic peptides, reviewed (Plank C, Zauner W, Wagner E: Application of membrane-active peptides for drug and gene delivery across cellular membranes, Advanced Drug Delivery Reviews, 1998, 34, 21-35. Wagner E, Effects of membrane-active agents in gene delivery, J. Cont. Release, 1998, 53, 155-158.). Polymers, some with zwitterionic features, (Richardson S, Kolbe H, Duncan R: Potential of low molecular mass chitosan as a DNA delivery system: Biocompatibility, body distribution and ability to complex and protect DNA Int. J. Pharm., 1999 178, 231-243. Richardson S, Ferruti P, Duncan R: Poly(amidoamine)s as potential endosomolytic polymers: Evaluation of body distribution in normal and tumour baring animals, J. Drug Targeting, 1999) have been shown to have considerable potential for membranelytic activity as a function of pH which could be capable of rupturingthe endosome to gain access to the ctyosolic environment of cells.
For the treatment of cancer there are marked improvements in therapeutic efficacy and site specific passive capture through the enhanced permeability and retention (EPR) effect (Matsumura Y, Maeda H: A new concept for macromolecular therapeutics in cancer chemotherapy; mechanism of tumoritropic accumulation of proteins and the antitumour agent SMANCS. Cancer Res., 1986, 6, 6387-6392.). The EPR effect results from enhanced permeability of macromolecules or small particles within the tumour neovasculature due to leakiness of its discontinuous endothelium. In addition to the tumour angiogenesis (hypervasculature) and irregular and incompleteness of vascular networks, the attendant lack of lymphatic drainage promotes accumulation of macromolecules that extravasate. This effect is observed in many solid tumours for macromolecular agents and lipids. The enhanced vascular permeability will support the demand of nutrients and oxygen for the unregulated growth of the tumour. Unless specifically addressed for tumour cell uptake by receptor-medicated endocytosis, polymers entering the intratumoural environment are taken up relatively slowly by fluid-phase pinocytosis. Whereas cellular uptake of low molecular weight molecules usually occurs by rapid transmembrane passage, the uptake of pysiologically soluble polymers occurs almost exclusively by endocytosis (Mellman I: Endocytosis and molecular sorting. Ann. Rev. Cell Develop. Biol., 1996, 12, 575-625. Duncan R, Pratten M: Pinocytosis: Mechanism and Regulation. In: Dean R, Jessup W, eds. Mononuclear Phagocytes: Physiology and Pathology. Amsterdam: Elsevier Biomedical Press, 1985; 27-51.).
Polymer bioactive conjugates can additionally include a conjugated bioactive agent that would induce receptor-mediated endocytosis (Putnam D, Kopecek J: Polymer conjugates with anticancer activity. Adv.Polym.Sci., 1995, 122, 55-123. Duncan R: Drug-polymer conjugates: potential for improved chemotherapy. Anti-Cancer Drugs, 1992, 3, 175-210.). For example, HPMA copolymer-doxorubicin containing additionally galactosamine localises selectively in the liver due to uptake by the hepatocyte asialoglycoprotein receptor (Duncan R, Seymour L, Scarlett L, Lloyd J, Rejmanova P, Kopecek J: N-(2-Hydroxypropyl)methacrylamide copolymers with pendant galactosamine residues. Fate after intravenous administration to rats. Biochim. Biophys. Acta., 1986, 880, 62-71. Seymour L, Ulbrich K, Wedge S, Hume I, Strohalm J, Duncan R: N-(2hydroxypropyl)methacrylamide copolymers targeted to the hepatocyte galactose-receptor: pharmacokinetics in DBA-2 mice. Br. J. Cancer, 1991, 63, 859-866.).
Enhanced vascular permeability is well known to be present within tissue which has undergone an inflammatory response due to infection or autoimmunedisease. Conjugates of polymers and appropriate bioactive agents could also exploit the vascular premeability gradient between healthy and inflammed tissue in these conditions leading to the passive and preferential accumulation of the conjugate at the inflammed site similar to that observed which has been shown at tumour sites in cancer.
Polymer bioactive conjugates designed to be therapeuctically efficacious by multivalent interactions are being developed as agonists, partial agonists, inverse agonists and antagonists for a multitude of clinical applications including the treatment of diseases such as cancer and infection (Griffin JH, Judice JK: Novel multi-binding therapeutic agents that modulate enzymatic processes, WO 99/64037. Yang G, Meier-Davis S, Griffin JH:Multivalent agonists, partial agonists, inverse agonists and antagonists of the 5-HT3 receptors, WO 99/64046. Christensen BG, Natarajan M, Griffin JH: Multibinding bradykinin antagonists, WO 99/64039. Fatheree P. Pace JL, Judice JK, Griffin JH: Preparation of multibinding Type II topoisomerase inhibitors as antibacterial agents, WO 99/64051. Linsell MS, Meier-Davis S, Griffin JH: Multibinding inhibitors of topoisomerase, WO 99/64054. Griffin JH, Moran EJ, Oare D: Novel therapeutic agents for macromolecular structures. PCT Int. Appl. WO 9964036. Griffin JH, Judice JK: Linked polyene macrolide antibiotic compounds and uses, WO 99/64040. Choi S, Mammen M, Whitesides GM, Griffin JH: Polyvalent presenter combinatorial libraries and their uses, WO 98/47002.).
The four main parts of a polymer-bioactive agent conjugate are (1) polymer, (2) bioactive agent conjugating linker which can be either a pendent chain conjugating linker or a mainchain terminating conjugating linker, (3) solution solubilising pendent chain and (4) the conjugated bioactive agent. While each component has a defined biological function, the sum is greater than the parts because these four components together as a conjugate produce a distinct profile of pharmacological, pharmacokinetic and physicochemical properties typical of physiologically soluble polymer-bioactive agent conjugates. The polymer is not a mere carrier for the bioactive agent. The polymer component of the conjugate can be synthetic or naturally derived. Synthetically derived polymers have the advantage that structure property correlations can be more effectively modulated and correlated in unique ways (Brocchini S, James K, Tangpasuthadol V, Kohn J: Structure-property correlations in a combinatorial library of degradable biomaterials. J. Biomed. Mater. Res., 1998, 42(1), 66-75. Brocchini S, James K, Tangpasuthadol V, Kohn J: A Combinatorial Approach For Polymer Design. J. Am. Chem. Soc., 1997, 119(19), 4553-4554.).
The solution properties of the polymer are directly responsible for defining the circulation half-life, rate of cellular uptake, minimising deleterious side effects of potent cytotoxic drugs and imparting favourable physicochemical properties (e.g. increasing the solubility of lipophilic drugs). The solution properties of a polymer bioactive agent conjugate will be influenced by the structure of the polymer, the conjugating linker and the property modifying pendent chain. Also the amount or loading of the bioactive agent will affect the solution properties of a polymer bioactive conjugate. The solution properties of the conjugate will affect the ultimate biological profile of the conjugate.
Solution properties will contribute to the biocompatibility and rate of clearance of polymer bioactive agent conjugates. Biocompatibility includes the lack of conjugate binding to blood proteins and the lack of a immunogenic response. The conjugate will display a plasma clearance which is primarily governed by the rate of kidney glomerular filtration and the rate of liver uptake. Macromolecules of molecular weight of 40,000-70,000 Da, depending on solution structure, readily pass through the kidney glomerulus and can be excreted. However, as the solution size of a molecule increases with molecular weight (or by forming supramolecular aggregates), extended blood clearance times result. Structural features including polymer flexibility, charge, and hydrophobicity affect the renal excretion threshold for macromolecules within this size range (Duncan R, Cable H, Rypacek F, Drobnik J, Lloyd J: Characterization of the adsorptive pinocytic capture of a polyaspartamide modified by the incorporation of tyramine residues. Biochim. Biophys. Acta, 1985, 840, 291-293.). Neutral, hydrophilic polymers including HPMA copolymers, polyvinylpyrrolidone (PVP) and poly(ethylene glycol) (PEG) have flexible, loosely coiled solution structures whereas proteins tend to be charged and exhibit more compact solution structures. For example, the molecular weight threshold limiting glomerular filtration of HPMA copolymer-tyrosinamide in the rat was approximately 45,000 Da (Seymour L, Duncan R, Strohalm J, Kopecek J: Effect of molecular weight (Mw) of N-(2-hydroxypropyl)methacrylamide copolymers on body distributions and rate of excretion after subcutaneous, intraperitoneal and intravenous administration to rats. J. Biomed. Mater. Res., 1987, 21, 1341-1358.) and the threshold for proteins is approximately 60K Da.
Copolymers HPMA have been extensively studied for the conjugation of cytotoxic drugs for cancer chemotherapy (Duncan R, Dimitrijevic S, Evagorou E: The role of polymer conjugates in the diagnosis and treatment of cancer. STP Pharma, 1996, 6, 237-263. Putnam D, Kopecek J: Polymer conjugates with anticancer activity. Adv.Polym.Sci., 1995, 122, 55-123. Duncan R: Drugolymer conjugates: potential for improved chemotherapy. Anti-Cancer Drugs, 1992, 3, 175-210.). The homopolymer of HPMA is soluble in biological fluids, readily excreted at molecular weights of less than 40,000 Da [4], is non-toxic up to 30 glkg, does not bind blood proteins [5], and is not immunogenic (Rihova B, Ulbrich K, Kopecek J, Mancal P: Immunogenicity of N-(2-hydroxypropyl)methacrylamide copolymers-potential hapten or drug carriers. Folia Microbioa., 1983, 28, 217-297. Rihova B, Kopecek J, Ulbrich K, Chytry V: Immunogenicity of N-(2-hydroxypropyl)methacrylamide copolymers. Makromol. Chem. Suppl., 1985, 9, 13-24. Rihova B, Riha I: Immunological problems of polymer-bound drugs. CRC Crit. Rev. Therap. Drug Carrier Sys., 1985, 1, 311-374. Rihova B, Ulbrich K, Strohalm J, Vetvicka V, Bilej M, Duncan R, Kopecek J: Biocompatibility of N-(2-hydroxypropyl)methacrylamide copolymers containing adriamycin. Immunogenicity, effect of haematopoietic stem cells in bone marrow in viva and effect on mouse splenocytes and human peripheral blood lymphocytes in vitro. Biomaterials, 1989, 10, 335-342.) Like poly(ethylene glycol) (PEG) which is generally recognised as safe (GRAS) and is used for the conjugation of proteins, HPMA is biocompatible and is thus a good candidate polymer for conjugation with bioactive agents. Since HPMA copolymers are hydrophilic, solublisation of hydrophobic drugs is possible. Since each HPMA copolymer conjugate is a different copolymer, other hydrophilic polymers similar to HPMA may be good candidate polymers for the conjugation of bioactive agents.
Additionally, the molecular weight characteristics of a polymer-bioactive agent conjugate will influence the ultimate biological profile of the conjugate. Biodistribution and pharmacological activity are known to be molecular weight-dependent. For example, blood circulation half-life (Cartlidge S, Duncan R. Lloyd J, Kopeckova-Rejmanova P, Kopecek J: Soluble crosslinked N-(2-hydroxypropyl)methacrylamide copolymers as potential drug carriers. 2. Effect of molecular weight on blood clearance and body distribution in the rat intravenous administration. Distribution of unfractionated copolymer after intraperitoneal subcutaneous and oral administration. J Con. Rel., 1986, 4, 253-264.), renal clearance, deposition in organs (Sprincl L, Exner J, Sterba 0, Kopecek J: New types of synthetic infusion solutions III. Elimination and retention of poly[N-(2-hydroxypropyl)methacrylamide] in a test organism. J. Biomed. Mater. Res., 1976, 10, 953-963.), rates of endocytic uptake (Duncan R. Pratten M, Cable H, Ringsdorf H, Lloyd J: Effect of molecular size of 125l-labelled poly(vinylpyrrolidone) on its pinocytosis by rat visceral yolk sacs and peritoneal macrophages. Biochem. J., 1981, 196, 49-55. Cartlidge S, Duncan R, Lloyd J, Rejmanova P, Kopecek J: Soluble crosslinked N2-hydroxypropyl)methacrylamide copolymers as potential drug carriers. 1. Pinocytosis by rat visceral yolk sacs and rat intestinal cultured in vitro. Effect of molecular weight on uptake and intracellular degradation. J. Cont. Rel., 1986, 3, 55-66.) and biological activity can depend on polymer molecular weight characteristics (Kaplan A: Antitumor activity of synthetic polyanion. In: Donaruma L, Ottenbrite R. Vogl O, eds. Anionic Polymeric Drugs. New York: Wiley, 1980; 227-254. Ottenbrite R, Regelson W, Kaplan A, Carchman R, Morahan P, Munson A: Biological activity of poly(carboxylic acid) polymers. In: Donaruma L, Vogi O, eds. Polymeric Drugs. New York: Academic Press, 1978; 263-304. Butler G: Synthesis, characterization, and biological activity of pyran copolymers. In: Donaruma L, Ottenbrite R, Vogl O, eds. Anionic Polymeric Drugs. New York: Wiley, 1980; 49-142. Muck K, Rolly H, Burg K: Makromol. Chem., 1977, 178, 2773. Muck K, Christ O, Keller H: Makromol. Chem., 1977, 178, 2785. Seymour L: Synthetic polymers with intrinsic anticancer activity. J. Bioact. Compat. Polymers, 1991, 6, 178-216.).
In clinical applications requiring the cellular uptake of a polymeric bioactive agent conjugate with subsequent release of the bioactive agent intracellularly, the linker must be designed to be degraded to release the bioactive agent at an optimal rate within the cell. It is preferable that a the bioactive agent conjugating linker does not degrade in plasma and serum (Vasey P, Duncan R, Twelves C, Kaye S, Strolin-Benedetti M, Cassidy J: Clinical and pharmacokinetic phase 1 study of PK1(HPMA) copolymer doxorubicin. Annals of Oncology, 1996, 7, 97.). Upon endocytic uptake into the cell, the conjugate will localise in the lysosomes. These cellular organalles contain a vast array of hydrolytic enzymes including proteases, esterases, glycosidases, phosphates and nucleases. For the treatment of cancer, potent cytotoxic drugs have been conjugated to polymers using conjugation linkers that degrade in the lysosome while remaining intact in the bloodstream. Since many drugs are not pharmacologically active while conjugated to a polymer, this results in drastically reduced toxicity compared to the free drug in circulation.
The conjugating linker structure must be optimised for optimal biological activity. Incorporation of a polymer-drug linker that will only release drug at the target site can reduce peak plasma concentrations thus reducing drug-medicated toxicity. If the drug release rate is optimised, exposure at the target can be tailored to suit the mechanism of action of the bioactive agent being used (e.g. use of cell-cycle dependent antitumour agents) and to prevent the induction of resistance. To be effective, it is important that polymer bioactive agent conjugates are designed to improve localisation of the bioactive agent in the target tissue, diminish deleterious exposure in potential sites of toxicity in other tissue and to optimise the release rate of the bioactive agent in those applications where its release is required for a biological effect. The rate of drug release from the polymer chain can also vary according to the polymer molecular weight and the amount of drug conjugated to the polymer. As greater amounts of hydrophobic drug are conjugated onto a hydrophilic polymer, the possibility to form polymeric micelles increases (Ulbrich K, Konak C, Tuzar Z, Kopecek J: Solution properties of drug carriers based on poly[N-(2hydroxypropyl)methacrylamide] containing biodegradable bonds. Makromol. Chem., 1987, 188, 1261-1272.). Micellar conjugate structures may hinder access of the lysosomal enzymes to degrade the linker and release the conjugated drug. Additionally, hydrophilic polymers conjugated to hydrophobic drugs can exhibit a lower critical solution temperature (LCST) where phase separation occurs and the conjugate becomes insoluble. Simple turbidometric assays (Chytry V, Netopilik M, Bohdanecky M, Ulbrich K: Phase transition parameters of potential thermosensitive drug release systems based on polymers of N-alkylmethacrylamides. J. Biomater. Sci. Polymer Ed., 1997, 8(11), 817-824.) have been used as a preliminary screen to determine the propensity for phase separation at various HPMA copolymer-doxorubicin conjugates of different molecular weight and drug loading (Uchegbu F, Ringsdorf H, Duncan R: The Lower Critical Solution Temperature of Doxorubicin Polymer Conjugates. Proceed. Intern. Symp. Control. Rel. Bioact. Mater., 1996.). As a bioactive agent is released from a polymer due to linker degradation it would be expected that changes in polymer conformation will occur that might also lead to diffences in drug release rate with time (Pitt C, Wertheim J, Wang C, Shah S: Polymer-drug conjugates: Manipulation of drug delivery kinetics. Macromol. Symp., 1997, 123, 225-234. Shah S, Werthim J, Wang C, Pitt C: Polymer-drug conjugates: manipulating drug delivery kinetics using model LCST systems. J. Cont. Rel., 1997, 45, 95-101.)and therefore pharmacological properties. The extent of drug loading and its influence on polymer solution properties is an important, and yet poorly understood phenomenon which must be correlated to structure-property relationships of the polymer-bioactive agent conjugate to lead to optimisation of the the in viva biological properties of therapeutic polymer bioactive agent conjugates. Currently HPMA copolymer-drug conjugates are prepared by a polymer analogous reaction of a low molecular weight drug (e.g. doxorubicin) with a copolymeric precursor which incorporates both the bioactive agent conjugating linker and the solution solubilising pendent chain (Rihova B, Ulbrich K, Strohalm J, Vetvicka V, Bilej M, Duncan R, Kopecek J: Biocompatibility of N-(2-hydroxypropyl)methacrylamide copolymers containing adriamycin. Immunogenicity, effect of haematopoietic stem cells in bone marrow in viva and effect on mouse splenocytes and human peripheral blood lymphocytes in vitro. Biomaterials, 1989, 10, 335-342. Kopecek J, Bazilova H: Poly[N-(hydroxypropyl)methecrylamide]-I. Radical polymerisation and copolymerisation. Eur. Polymer J., 1973, 9, 7-14. Strohalm J, Kopecek J: Poly[N-(2-hydroxypropyl)methacrylamide] IV. Heterogeneous polymerisation. Angew. Makromol. Chem., 1978, 70, 109-118. Rejmanova P, Labsky J, Kopecek J: Aminolyses of monomeric and polymeric 4-nitrophenyl esters of N-methacryloylamino acids. Makromol. Chem., 1977, 178, 2159-2168. Kopecek J: Reactive copolymers of N-(2-Hydroxypropyl)methacrylamide with N-methacryloylated derivatives of L-leucine and L-phenylalanine. Makromol. Chem., 1977, 178, 2169-2183. Kopecek J: The potential of water-soluble polymeric carriers in targeted and site-specific drug delivery. J. Cont. Rel., 1990, 11, 279-290.). The vast majority of polymer bioactive agent conjugates prepared by the polymer analogous reaction are prepared by the reaction of the bioactive agent with a copolymeric precursor (Note references in Brocchini S and Duncan R: Polymer drug conjugates: drug release from pendent linkers. The Encyclopedia of Controlled Drug Delivery, Wiley, N.Y., 1999, 786-816.).
The disadvantage of using a copolymer precursor is that for each change in the structure or relative amounts of (1) the bioactive agent conjugating linker or (2) the solution solubilising pendent chain, a new copolymeric precursor must be prepared. Since pendent chain structure is important for the biological profile of a polymer bioactive agent a copolymeric precursor is required to study each conjugate possessing modified conjugating linkers. Solution structure is a function of all the structural features of a bioactive agent polymer conjugate. To elucidate the solution-structure correlations of either the polymer mainchain, conjugating linker or solution solubilising pendent chain requires a different copolymer precursor for each variation of each component.
It is not possible to even use the same copolymeric precursor to vary the amount or loading of the conjugated bioactive agent. If loading of the bioactive agent is to be varied and is to be less than the relative stoichiometry of the conjugating pendent chain, then the remaining conjugating pendent chains will not be conjugated to a drug, and the remaining conjugating pendent chains will be terminate with some other inert molecule. The polymer analogous reaction requires that the copolymeric precursor possess functionality on the conjugation pendent chain termini that is reactive (e.g. a p-nitrophenol active ester of a carboxylic acid) so that upon addition of a bioactive agent, the agent will form a covalent bond with the conjugation pendent chain to become linked to the polymer. Thus if a loading of the bioactive agent is to be less than the relative stoichiometry of the conjugating pendent chain, the reactive functionality must be quenched with a reagent other than the bioactive agent or preferably in this situation, a new polymeric precursor be prepared. These procedures tend to produce polymer conjugates with a wide distribution of structures. It thus becomes impossible to accurately determine structure-property correlations. Clearly, if a loading of the bioactive agent greater than the relative stoichiometry of the conjugation pendent chain is desired, then another copolymeric precursor must be prepared.
Since many polymer bioactive agent conjugates are co-poly-(methacrylamides), the polymer analogous reaction is conducted on a co-poly(methacrylamide) precursor. It is not possible to make the vast majority of such precursors with a narrow molecular weight distribution with a polydisperisty index of less than 2 except in special cases where a copolymer precursor happens to precipitate from the polymerisation solution at a molecular weight below the renal threshold. It is also not possible to make several different copoly(methacrylamides) all possessing the same molecular weight characteristics, e.g. all possessing the same degree of polymerisation and the same molecular weight distribution. The copoly-(methacrylamide) precursors tend to be prepared by free radical polymerisation which typically produce random copolymers typically with a polydisperisity (PD) greater than 1.5-2.0.
Furthermore since the relative stoichiometry of the conjugated bioactive agent, and thus the conjugating linker, is less than the solution solubilising pendent chain, the polymer analogous reaction is frequently on a copolymer precuresor with a low relative stoichiometry of reactive sites for the conjugation of the bioactive agent. This inefficient conjugation strategy is often burdened with competitive hydrolysis reactions and other consuming side reaction that result in conjugating linkers not covalently linked to the bioactive agent (Mendichi R, Rizzo V, Gigli M, Schieroni A G: Molecular characterisation of polymeric antitumour drug carriers by size exclusion chromatograpgy and universal calibration. J. Liq. Chrom. and Rel. Technol., 1996, 19(10), 1591-1605. Configliacchi E, Razzano G, Rizzo V, Vigevani A: HPLC methods for the determination of bound and free doxorubicin and of bound and free galactosamine in methacrylamide polymer-drug conjugates. J. Pharm. Biomed. Analysis, 1996, 15, 123-129.). This not only causes significant structure heterogenaity between batches, but also causes significant waste of the bioactive agent because it has not been conjuated and its recovery is too expensive. In the case of conjugate developed for endocytic uptake into a cell, the lysosomal degradation of bioactive agent conjugating pendent chains with pendent chains not linked to the bioactive pendent chain. This competition complicates the pharmacology and pharmacokinetics of the polymer bioactive agent conjugate.
Polymer-bioactive agent conjugates and biomedical polymers currently used for medical applications are, from the perspective of regulatory agencies (e.g. Medicines Control Agency, FDA) not structurally defined. Many conjugates display broad molecular weight distribution and random incorporation of the conjugated bioactive agent. Frequently, the structure of the conjugating linker is varied due to racimisation or incomplete conjugation of the bioactive agent to each of the conjugating linkers.
Future development of physiologically soluble polymers used in the development of polymer-bioactive agent conjugates (i.e. polymer therapeutics) requires that more defined conjugate structures be prepared for study. In this way it will become possible to more accurately elucidate structure-property correlations that influence the biological profile of these macromolecular therapeutics. This is not possible by conducting the polymer analogous reaction on many different copolymeric precursors. There is a need to prepare polymer-bioactive conjugates which have a more narrow molecular weight distribution than are currently available. There is also a need to ensure that each bioactive conjugating linker is structurally the same and is covalently bound to the polymer and the bioactive agent. Additionally there is a need for a more efficient strategy in preclinical development where conjugates with similar molecular weight characteristics are prepared for study and where solution properties can also be varied without changing the molecular weight characteristics of the polymer mainchain. Since HPMA copolymer conjugates are poly(methacrylamides) then any techniques developed that will meet the requirements to prepare such conjugates can also be used to prepare other poly(methacrylates) for other healthcare and consumer applications where the resultant polymer can be used either as a soluble molecule, processible material that can be fabricated into a device or as an excipient. Since only a small limited number of acrylamide homo- and co-polymers with narrow molecular weight distribution can be prepared, then for speciality applications there is need for processes that provide a means to prepare such polymers.
These limitations for conducting the polymer analogous reaction on a copolymer precursor can be alleviated by conducting the polymer analogous reaction with a homopolymeric precursor that has a narrow molecular weight distribution and where each repeat unit is reactive site. Conjugation of a bioactive agent or a derivative is carried out in a first reaction to covalently link the bioactive agent to the polymer. The conjugation is efficient because each repeat unit on the homopolymer precursor is a reactive site available for reaction. Upon conjugation of the bioactive agent, the intermediate precursor is a copolymer comprised of most repeat units being terminated still with a reactive chemical functional group. These are are then allowed to react with a reagent which will become the solution solubilising pendent chain in the final conjugate. By using one such narrow molecular weight distribution homopolymeric precursor it becomes possible to prepare many copolymer conjugates all possess the same narrow molecular weight distribution. Each conjugate will also possess the same molecular weight characteristics of the degree of polymerisation and polydispersity index that the homopolymeric percursor possesses.
This invention is concerned with the synthesis by controlled radical polymerisation processes (Sawamoto M, Masami K: Living radical polymerizations based on transition metal complexes. Trends Polym. Sci. 1996, 4, 371-377. Matyjaszewski K:, Mechanistic and synthetic aspects of atom transfer radical polymerization. Pure Appl. Chem. 1997, A34, 1785-1801. Chiefari J, Chong Y, Ercole F, Krstina J, Jeffery J, Le T, Mayadunne R, meijs G, Moad C, Moad G, Rizzardo E, Thang S: Living free-radical, polymerization by reversible addition-fragmentation chain transfer: the RAFT process. Macromolecules, 1998, 31, 5559-5562. Benoit D, Chaplinski V, Braslau R. Hawker C: Development of a universal alkoxyamine for xe2x80x9clivingxe2x80x9d free radical polymerizations. J. Am. Chem. Soc., 1999, 121, 3904-3920.) of narrow molecular weight distribution homopolymer precursors with a polydispersity index of less than 1.2. These controlled radical polymerisation processes have so far not been shown to give directly acrylamide homo- and co-polymers with narrow molecular weight distribution. This invention is also concerned with the use of these homopolymeric precursors to prepare physiologically soluble polymer bioactive agent conjugates, polymer therapeutics, functionalised polymers, pharmaceutical compositions and materials.
One embodiment of the present invention provides a polymer comprising the unit (I) 
wherein R is selected from the group consisting of hydrogen, C1-C18 alkyl, C1-C18 alkenyl, C1-C18 aralkyl, C1-C18 alkaryl, carboxylic acid, carboxy-C1-6 alkyl, or any one of C1-C18 alkyl, C1-C18 alkenyl, C1-C18 aralkyl, C1-C18 alkaryl substituted with a heteroatom within, or attached to, the carbon backbone; R1 is selected from the group consisting of hydrogen, C1-C6 alkyl groups; X is an acylating group and wherein the polymer has a polydispersity of less than 1.4, preferably less than 1.2 and a molecular weight (Mw) of less than 100,000.
The acylating group X is preferably a carboxylate activating group and is generally selected from the group consisting of N-succinimidyl, pentachlorophenyl, pentafluorophenyl, para-nitrophenyl, dinitrophenyl, N-phthalimido, N-norbornyl, cyanomethyl, pyridyl, trichlorotriazine, 5-chloroquinilino, and imidazole. Preferably X is an N-succinimidyl or imidazole moiety.
Preferably R is selected from the group consisting of hydrogen, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 aralkyl and C1-C6 alkaryl, C1-C6 alkylamido and C1-C6 alkylamido. Most preferably R is selected from hydrogen or methyl.
Preferably R1 is selected from the group consisting of hydrogen, methyl, ethyl, propyl, butyl, pentyl or isomers thereof. Most preferably R1 is selected from hydrogen or methyl.
The polymer of the present invention may be a homopolymer incorporating unit (I), or may be a copolymer or block copolymer incorporating other polymeric, oligomeric or monomeric units. For example, further polymeric units incorporated in the polymer may comprise acrylic polymers, alkylene polymers, urethane polymers, amide polymers, polypeptides, polysaccharides and ester polymers. Preferably, where the polymer is a heteropolymer, additional polymeric components comprise polyethylene glycol, polyaconitic acid or polyesters.
The molecular weight of the polymer should ideally be less than 100,000, preferably 50,000 where the polymer is to be used as a physiologically soluble polymer (in order that the renal threshold is not exceeded). Preferably the molecular weight of the polymer is in the range of 50,000-4000, more preferably 25,000-40,000. Another embodiment of the present invention is a polymer comprising the unit (II) 
wherein R2 is selected from hydrogen, C1-C18 alkyl, C1-C18 alkenyl, C1-C18 aralkyl, C1-C18 alkaryl, carboxylic acid and carboxy-C1-6alkyl; R3 is selected from the group consisting of hydrogen, methyl, ethyl, propyl, butyl, pentyl and isomers thereof, Z is a pendent group selected from the group consisting of NR4R5, SR6 and OR7, wherein R4 is an acyl group, preferably an aminoacyl group or oligopeptidyl group; R5 is selected from hydrogen, C1-C18 alkyl, C1-C18 alkenyl, C1-C18 aralkyl, C1-C18 alkaryl; R6 and R7 are selected from the group consisting of hydrogen, C1C12 alkyl, C1-C12 alkenyl, C1-C12 aralkyl, C1-C12 alkaryl, C1-C12 alkoxy and C1-C12 hydroxyalkyl, and may contain one or more cleavable bonds and may be covalently linked to a bioactive agent. Generally the polymer has a polydispersity of less than 1.4, preferably less than 1.2 and a molecular weight (Mw) of less than 100,000, preferably 50,000.
Preferably R2 is selected from the group consisting of hydrogen, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 aralkyl and C1-C6 alkaryl, C1-C6 alkylamido and C1-C6 alkylamido.
Preferably R3 is selected from the group consisting of hydrogen, methyl, ethyl, propyl, butyl, pentyl or isomers thereof. Most preferably R2 is hydrogen and R3 is hydrogen or methyl.
Z may comprise a peptidic group. Preferably Z comprises one or more aminoacyl groups, preferably 2-6 aminoacyl groups, most preferably 4 aminoacyl groups. In a particularly preferred embodiment group Z comprises a glycine-leucine-phenylalanine-glycine linkage. The aminoacyl linkage is most preferably a peptide linkage capable of being cleaved by preselected cellular enzymes, for instance, those found in liposome of cancerous cells. In another preferred embodiment group Z comprises a cis-aconityl group. This group is designed to remain stable in plasma at neutral pH (xcx9c7.4), but degrade intracellularly by hydrolysis in the more acidic environment of the endosome or liposome (xcx9cpH 5.5-6.5).
The pendent chain Z may additionally be covalently bound to a ligand or bioactive agent. The ligand may be any ligand which is capable of polyvalent interactions. Preferred bioactive agents are anti-cancer agents such as doxorubicin, daunomycin and paclaxitol.
A further preferred polymer of the present invention has the structure (III) 
wherein R8 and R9 are selected from the same groups as R2 and R3 respectively, Q is a solubilising groups selected from the group consisting of C1C12 alkyl, C1-C12 alkenyl, C1-C12 aralkyl, C1 -C12 alkaryl, C1-C12 alkoxy, C1-C12 hydroxyalkyl, C1-C12 alkylamido, C1-C12 alkylamido, C1-C12 alkanoyl, and wherein p is an integer of less than 500.
Preferably Q comprises an amine group, preferably a C1-C12 hydroxyalkylamino group, most preferably a 2-hydroxypropylamino moiety. This group is designed to be a solubilising group for the polymer in aqueous solutions. Generally the polymer of the present invention is a water soluble polyacrylamide homo- or copolymer, preferably a polymethacrylamide or polyethacrylamide homo- or copolymer.
In a further embodiment, the present invention provides a process for the production of a polymer, comprising the polymerisation of a compound (IV) 
wherein R is selected from the group consisting of hydrogen, C1-C18 alkyl, C1-C18 alkenyl, C1-C18 aralkyl, C1-C18 alkaryl, carboxylic acid, carboxy-C1-6alkyl, or any one of C1-C18 alkyl, C1-C18 alkenyl, C1-C18 aralkyl, C1-C18 alkaryl substituted with a heteroatom within, or attached to, the carbon backbone; R1 is selected from the group consisting of hydrogen and C1-C6 alkyl groups preferably selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl and isomers thereof; X is an acylating group, preferably a carboxylate activating group; wherein the process is a controlled radical polymerization, to produce a narrow weight distribution polymer comprising the unit (I) 
wherein n is an integer of 1 to 500.
Preferably R is selected from the group consisting of hydrogen, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 aralkyl and C1-C6 alkaryl, C1-C6 alkanoyl, C1-C6 alkylamido and C1-C6 alkylamido.
Preferably R1 is selected from the group consisting of hydrogen, methyl, ethyl, propyl, butyl, pentyl or isomers thereof.
Where the polymerization is carried out by atom transfer radical polymerization, a suitable radical initiator is utilised. Such initiators commonly comprise alkylhalides, preferably alkylbromides. In particular, the initiator is 2-bromo-2-methyl-(2-hydroxyethyl)propanoate. The polymerisation is also carried out in the presence of a polymerisation mediator comprising a Cu(I) complex. Such complexes are usually Cu(I)Br complexes, complexed by a chelating ligand. Typical mediators are Cu(I)Br (Bipy)2, Cu(I)Br(Bipy), Cu(I)Br(Pentamethyl diethylene), Cu(I)Br[methyl6 tris(2-aminoethyl)amine] and Cu(I)Br(N,N,Nxe2x80x2,Nxe2x80x3,Nxe2x80x3-pentamethyidiethylenetriamine).
The reaction should take place in the presence of a suitable solvent. Such solvents are generally aprotic solvents, for example tetrahydrofuran, acetonitrile, dimethylformamide, acetone, dimethylsulphoxide, ethyl acetate, methylformamide and sulpholane and mixtures thereof. Alternatively, water may be used. Particularly preferred solvents are dimethylsulphoxide and dimethylformamide and mixtures thereof.
Alternatively the polymerization may take place via Nitroxide Mediated Polymerization. Again, a suitable Nitroxide Mediated Polymerization initiator is required. Such an initiator generally has the structure 
wherein A is selected from the group consisting of C1-C12 alkyl, C1-C12 alkenyl, C1-C12 aralkyl, C1-C12 alkaryl, C1-C12 hydroxyalkyl, B and C are individually selected from the group consisting of C1C12 alkyl, C1-C12 alkenyl, C1-C12 aralkyl, C1-C12 alkaryl, C1-C12 hydroxyalkyl, and may together with N form a C1-C12 heterocyclic group and which may contain a heteroatom selected from nitrogen, sulfur, oxygen and phosphorus.
Preferably A is selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, hexyl, benzyl, methylbenzene, ethyl benzene, propylbenzene or isomers thereof. B and C should generally be sterically crowding the groups capable of stabilising a nitroxide radical. Such groups are generally selected from the group consisting of isopropyl, isobutyl, secbutyl, tert-butyl, isopentyl, sec-pentyl, tert-pentyl, adamantyl, methylbenzene, ethyl benzene, propylbenzene or isomers thereof.
Common initiators have these structures outlined below 
wherein R9 to R11 are selected from the group consisting of C1-C12 alkyl, C1-C12 alkenyl, C1-C12 aralkyl, C1-C12 alkaryl, C1-C12 alkoxy, C1-C12 hydroxyalkyl, C1-C12 alkylamido, C1-C12 alkylamido, C1-C12 alkanoyl.
Again, suitable solvents for use with Nitroxide Mediated Polymerisations are aprotic solvents as described above. Alternatively, water may be used. A further embodiment of the present invention provides a process for the production of a polymer, comprising the reaction of a polymer having the formula (VI) 
wherein R12 is a group selected from the group consisting of hydrogen, C1-C18 alkyl, C1-C18 alkenyl, C1-C18 aralkyl and C1-C18 alkaryl groups; R13 is selected from the group consisting of C1-C6 alkyl groups; E is a carboxylate activating group and r is an integer of 5 to 500; with a reagent HRx, wherein Rx is selected from the group consisting of NR14R15, SR16 and OR17, wherein R14 is an acyl group, preferably an aminoacyl group or oligopeptidyl group; R15 is selected from hydrogen, C1-C18 alkyl, C1-C18 alkenyl, C1-C18 aralkyl, C1-C18 alkaryl; R16 and R17 are selected from the group consisting of hydrogen, C1C12 alkyl, C1-C12 alkenyl, C1-C12 aralkyl, C1-C12 alkaryl, C1-C12 alkoxy and C1-C12 hydroxyalkyl, and may contain one or more cleavable bonds, to form a derivatised polymer having the structure (VII) 
wherein 1xe2x89xa6sxe2x89xa6r.
R12 is preferably selected from the group consisting of hydrogen, methyl, ethyl and propyl, R13 is selected from the group consisting of hydrogen, methyl, ethyl and propyl and preferably R12 is hydrogen and R13 are methyl. E is selected from the group consisting of N-succinimidyl, pentachlorophenyl, pentafluorophenyl, para-nitrophenyl, dinitrophenyl, N-phthalimido, N-norbornyl, cyanomethyl, pyridyl, trichlorotriazine, 5-chloroquinilino, and imidazole, preferably N-succinimidyl or imidazole, most preferably N-succinimidyl.
Preferably HRx is H2NR14.
HRx is generally a nucleophilic reagent capable of displacing Exe2x80x94O, to form a covalent bond with the acyl group attached to CR3. Preferably HRx comprises a primary or secondary amine group. Most preferably HRx comprises a cleavable bond such as a aminoacyl linkage or a cis-aconityl linkage as described hereinbefore. Generally HRx is covalently attached to a bioactive agent prior to reaction with (VI) subsequent to the production of a polymer having the structure (VII), an additional step of quenching the polymer may take place. This involves reacting the previously unreacted groups E with a quenching group. This group has the formula HRxxe2x80x2, preferably comprises an amine moiety and is generally selected to be a solubilising or solubility modifying group for the polymer. Such a quenching compound is, for example a hydrophilic reagent, for example, hydroxypropylamine.
The present invention provides a polymer having a polydispersity of less than 1.2. The polymer is preferably an activated polyacrylate ester that is prepared by Controlled Radical Polymerization. These polymers are designed to be derivitisable and may be used to form polymer-drug conjugates having improved biological profile. A particularly preferred polymer of the present invention comprises the structure (X) 
The activating moiety is an N-succinimidyl group. This particular group has been found to be particularly stable in solution and resists spontaneous hydrolysis. This polymer may be produced by Atom Transfer Polymerization using a Cu(I)Br(pentamethyidiethylene) mediator. The polymerization involved the reaction of a monomer (IX) with a suitable aprotic solvent. 
In one preferred embodiment the solvent is tetrahydrofuran. In another preferred embodiment the solvent is dimethylsulphoxide and optionally dimethylformamide in admixture thereof. The reaction is preferably carried out under a nitrogen atmosphere and at a temperature of 0-150xc2x0 C. A preferred temperature range is 30-80xc2x0 C., most preferably 50-70xc2x0 C. The polymer comprising the unit (X) may subsequently be derivatised. The carboxyl activating group may be substituted by a suitable nucleophilic reagent. In order to form polymer drug conjugates it is preferable to derivatise unit (X) with a pendant moiety. Such a moiety could comprise a aminoacyl linkage or a hydrolytically labile linkage as defined hereinbefore. Such a linkage can degrade when entering the lysosome of a diseased cell, thus releasing a drug or drug precursor directly to the target site. Preferably a pendent moiety comprises a Gly-Leu-Phe-Gly linkage or a cis aconityl linkage. Such a pendent linkage may be covalently attached to a drug prior to polymer derivitisation or may be capable of being derivatised subsequent of attachment of the pendent moiety to the polymer backbone, In a preferred embodiment the polymer comprising the unit (X) is reacted with less than 1 equivalent of a pendent group, thus only substituting a pre-specified number of N-succinimidyl moieties. This allows a second, quenching step, which substitutes the remaining N-succinimidyl groups with a solubilising group. Such a group aids in the solubilisation of the polymer in aqueous solutions such as biological fluids. A preferred quenching agent should comprise an amine, for example 2-hydroxypropylamine. An overview of a preferred reaction process is provided in scheme 1 below. In this particular example, the drug doxorubicin is attached to the polymer via a GLFG linkage. 
n is an integer in the range of 1 to 500 and m is the number equivalent of pendent moieties reacted with the activated polymer.
CRP processes are known to result in the presence of dormant initiating moieties at the chain ends of linear polymers. In particular the use of nitroxide mediated radical polymerization may be used to prepare narrow molecular weight distributed block copolymers. This allows more defined introduction of drug conjugating pendent chains in the polymer. Outlined in Scheme 2 is an example of this approach to prepare a block copolymer precursor using the CRP process known as nitroxide mediated polymerization (NMP). 
wherein x and y are the number equivalent of the pendent moiety and quenching group respectively.
Thus, polymeric precursors (XI) and (XIII) are designed to be used as polymeric precursors for polymer analogous reactions that are driven to completion to prepare conjugates with narrow molecular weight distributions and with differing m and n repeat structure. Drug conjugation would be localized only in the n repeat structure. Again it is possible to vary the solubilising pendent chain and the drug conjugating pendent chain starting from the polymeric precursor (XI). Defining the location of the drug conjugating pendent chains is necessary to develop more defined polymer-drug conjugates. The extent and location of drug loading and its influence on polymer solution properties is an important, and yet poorly understood phenomenon and will have a fundamental effect on the in vivo properties of therapeutic polymer-conjugates. Thus, this approach will find utility also in the development and optimization of polymer-drug conjugates.